microRNAs are negative and positive post-transcriptional regulators of BRAF-X1 mRNA [miRNA]

X1 is the most abundant transcript variant of BRAF mRNA and has a long 3’UTR, but its involvement in post-transcriptional regulatory circuits has not been investigated yet. Here, we describe 20 microRNAs that bind directly to the X1 3’UTR. They were identified in BRAFV600E mutant A375 melanoma cells using miR-CATCHv2.0, an implemented experimental method that combines RNA affinity purification with small RNA sequencing and an ad hoc analytical workflow. X1-targeting microRNAs fall into 4 classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and on the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). In many cases, the microRNA-induced variations in BRAFV600E protein levels are coupled to consistent variations in pMEK levels and, in turn, to melanoma cell proliferation and sensitivity to the BRAF inhibitor vemurafenib. However, examples exist of microRNAs that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein. Our study describes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells. The expression levels of microRNAs in A375 cells were measured by small RNA sequencing upon 48h of treatment with DMSO. Two independent replicates were analyzed.